U.S. patent application number 11/064065 was filed with the patent office on 2005-07-21 for aerodynamically light particles for pulmonary drug delivery.
Invention is credited to Ben-Jebria, Abdellaziz, Caponetti, Giovanni, Edwards, David A., Hanes, Justin, Hrkach, Jeffrey S., Langer, Robert S., Lotan, Noah.
Application Number | 20050158249 11/064065 |
Document ID | / |
Family ID | 27096992 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158249 |
Kind Code |
A1 |
Edwards, David A. ; et
al. |
July 21, 2005 |
Aerodynamically light particles for pulmonary drug delivery
Abstract
Improved aerodynamically light particles for drug delivery to
the pulmonary system, and methods for their synthesis and
administration are provided. In a preferred embodiment, the
aerodynamically light particles are made of biodegradable material
and have a tap density of less than 0.4 g/cm.sup.3 and a mass mean
diameter between 5 .mu.m and 30 .mu.m. The particles may be formed
of biodegradable materials such as biodegradable polymers. For
example, the particles may be formed of a functionalized polyester
graft copolymer consisting of a linear .alpha.-hydroxy-acid
polyester backbone having at least one amino acid group
incorporated therein and at least one poly(amino acid) side chain
extending from an amino acid group in the polyester backbone. In
one embodiment, aerodynamically light particles having a large mean
diameter, for example greater than 5 .mu.m, can be used for
enhanced delivery of a therapeutic agent to the alveolar region of
the lung. The aerodynamically light particles incorporating a
therapeutic agent may be effectively aerosolized for administration
to the respiratory tract to permit systemic or local delivery of
wide variety of therapeutic agents.
Inventors: |
Edwards, David A.; (Boston,
MA) ; Caponetti, Giovanni; (Piacenza, IT) ;
Hrkach, Jeffrey S.; (Cambridge, MA) ; Lotan,
Noah; (Haifa, IL) ; Hanes, Justin; (Baltimore,
MD) ; Ben-Jebria, Abdellaziz; (State College, PA)
; Langer, Robert S.; (Newton, MA) |
Correspondence
Address: |
ELMORE CRAIG & VANSTONE, P.C.
209 MAIN STREET
N. CHELMSFORD
MA
01863
US
|
Family ID: |
27096992 |
Appl. No.: |
11/064065 |
Filed: |
February 23, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11064065 |
Feb 23, 2005 |
|
|
|
10441948 |
May 20, 2003 |
|
|
|
10441948 |
May 20, 2003 |
|
|
|
10027212 |
Dec 20, 2001 |
|
|
|
6635283 |
|
|
|
|
10027212 |
Dec 20, 2001 |
|
|
|
09562988 |
May 1, 2000 |
|
|
|
6399102 |
|
|
|
|
09562988 |
May 1, 2000 |
|
|
|
09211940 |
Dec 15, 1998 |
|
|
|
6136295 |
|
|
|
|
09211940 |
Dec 15, 1998 |
|
|
|
08739308 |
Oct 29, 1996 |
|
|
|
5874064 |
|
|
|
|
08739308 |
Oct 29, 1996 |
|
|
|
08655570 |
May 24, 1996 |
|
|
|
Current U.S.
Class: |
424/46 ;
514/10.3; 514/10.4; 514/11.1; 514/11.8; 514/11.9; 514/171; 514/221;
514/305; 514/317; 514/343; 514/44R; 514/456; 514/5.9 |
Current CPC
Class: |
A61K 9/1647 20130101;
A61K 31/137 20130101; A61K 9/0075 20130101 |
Class at
Publication: |
424/046 ;
514/002; 514/015; 514/003; 514/171; 514/044; 514/343; 514/317;
514/221; 514/305; 514/456 |
International
Class: |
A61K 048/00; A61K
031/573; A61K 031/56; A61K 038/09; A61K 031/4439; A61K 031/353 |
Claims
What is claimed is:
1. A mass of biocompatible particles, suitable for pulmonary
delivery, comprising particles having therapeutic, prophylactic or
diagnostic agent, and having a mass mean diameter of at least 6.5
.mu.m and an aerodynamic diameter of less than 4 .mu.m, wherein at
least 46% of the mass of particles are deposited after the first
bifurcation of the lungs.
2. The particles of claim 1 wherein the particles have a mass mean
diameter between 6.5 .mu.m and 30 .mu.m.
3. The particles of claim 1 wherein the particles have an
aerodynamic diameter of less than 3 .mu.m.
4. The particles of claim 1 wherein the agent is selected from the
group consisting of proteins, polysaccharides, lipids, nucleic
acids, beta agonists and combinations thereof.
5. The particles of claim 1 wherein the agent is selected from the
group consisting of insulin, calcitonin, leuprolide, LHRH,
granulocyte colony stimulating factor, parathyroid hormone-related
peptide, somatostatin, testosterone, progesterone, estradiol,
nicotine, fentanyl, norethisterone, clonidine, scopolomine,
salicylate, cromolyn sodium, salmeterol, formeterol, albuterol, and
vallium.
6. The particles of claim 1 wherein the particles further comprise
a biodegradable material.
7. The particles of claim 6 wherein the biodegradable material is a
polymeric material.
8. The particles of claim 6 wherein the biodegradable material is a
non-polymeric material.
9. The particles of claim 1 wherein the particles have a tap
density of less than about 0.4 g/cm.sup.3.
10. The particles of claim 9 wherein the particles have a tap
density of less than about 0.1 g/cm.sup.3.
11. A method for delivery of a therapeutic, prophylactic or
diagnostic agent to the pulmonary system comprising: administering
to the respiratory tract of a patient in need of treatment,
prophylaxis or diagnosis an effective amount of biocompatible
particles, wherein the particles comprising a therapeutic,
prophylactic or diagnostic agent, and have a mass mean diameter of
at least 6.5 .mu.m and an aerodynamic diameter of less than 4
.mu.m, wherein 46% of the mass of said particles delivered are
deposited after the first bifurcation of the lungs.
12. The method of claim 11 wherein the particles have a mass mean
diameter between 6.5 .mu.m and 30 .mu.m.
13. The method of claim 11 wherein the particles have an
aerodynamic diameter of less than 3 .mu.m.
14. The method of claim 11 wherein the agent is selected from the
group consisting of proteins, polysaccharides, lipids, nucleic
acids, beta agonists and combinations thereof.
15. The method of claim 11 wherein the agent is selected from the
group consisting of insulin, calcitonin, leuprolide, LHRH,
granulocyte colony stimulating factor, parathyroid hormone-related
peptide, somatostatin, testosterone, progesterone, estradiol,
nicotine, fentanyl, norethisterone, clonidine, scopolomine,
salicylate, cromolyn sodium, salmeterol, formeterol, albuterol, and
vallium.
16. The method of claim 11 wherein the particles further comprise a
biodegradable material.
17. The method of claim 16 wherein the biodegradable material is a
polymeric material.
18. The method of claim 16 wherein the biodegradable material is a
non-polymeric material.
19. The method of claim 11 wherein the particles have a tap density
of less than about 0.4 g/cm.sup.3.
20. The method of claim 19 wherein the particles have a tap density
of less than about 0.1 g/cm.sup.3.
21. The method of claim 11 wherein the agent is delivered to the
deep lung.
22. The method of claim 11 wherein the agent is delivered to the
central airways.
23. A particulate composition suitable for pulmonary delivery of a
therapeutic, prophylactic or diagnostic agent, comprising particles
having a tap density less than 0.4 g/cm.sup.3, wherein more than
50% of the particles are respirable.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/441,948, filed May 20, 2003 which is a
continuation of Ser. No. 10/027,212, filed Dec. 20, 2001 which is a
continuation of co-pending U.S. patent application Ser. No.
09/562,988, filed May 1, 2000, which is a continuation of U.S.
patent application Ser. No. 09/211,940, filed Dec. 15, 1998, now
U.S. Pat. No. 6,136,295, which is a Divisional of U.S. application
Ser. No. 08/739,308 filed on Oct. 29, 1996, now U.S. Pat. No.
5,874,064 which is a continuation-in-part of U.S. patent
application Ser. No. 08/655,570, filed on May 24, 1996. The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to biodegradable
particles of low density and large size for drug delivery to the
pulmonary system.
[0003] Biodegradable particles have been developed for the
controlled-release and delivery of protein and peptide drugs.
Langer, R., Science, 249: 1527-1533 (1990). Examples include the
use of biodegradable particles for gene therapy (Mulligan, R. C.
Science, 260: 926-932 (1993)) and for `single-shot` immunization by
vaccine delivery (Eldridge et al., Mol. Immunol., 28: 287-294
(1991)).
[0004] Aerosols for the delivery of therapeutic agents to the
respiratory tract have been developed. Adjei, A. and Garren, J.
Pharm. Res. 7, 565-569 (1990); and Zanen, P. and Lamm, J. -W. J.
Int. J. Pharm. 114, 111-115 (1995). The respiratory tract
encompasses the upper airways, including the oropharynx and larynx,
followed by the lower airways, which include the trachea followed
by bifurcations into the bronchi and bronchioli. The upper and
lower airways are called the conducting airways. The terminal
bronchioli then divide into respiratory bronchioli which then lead
to the ultimate respiratory zone, the alveoli, or deep lung. Gonda,
I. "Aerosols for delivery of therapeutic and diagnostic agents to
the respiratory tract," in Critical Reviews in Therapeutic Drug
Carrier Systems 6:273-313, 1990. The deep lung, or alveoli, are the
primary target of inhaled therapeutic aerosols for systemic drug
delivery.
[0005] Inhaled aerosols have been used for the treatment of local
lung disorders including asthma and cystic fibrosis (Anderson et
al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have
potential for the systemic delivery of peptides and proteins as
well (Patton and Platz, Advanced Drug Delivery Reviews, 8:179-196
(1992)). However, pulmonary drug delivery strategies present many
difficulties for the delivery of macromolecules; these include
protein denaturation during aerosolization, excessive loss of
inhaled drug in the oropharyngeal cavity (often exceeding 80%),
poor control over the site of deposition, irreproducibility of
therapeutic results owing to variations in breathing patterns, the
often too-rapid absorption of drug potentially resulting in local
toxic effects, and phagocytosis by lung macrophages.
[0006] Considerable attention has been devoted to the design of
therapeutic aerosol inhalers to improve the efficiency of
inhalation therapies. Timsina et. al., Int. J. Pharm. 101, 1-13
(1995); and Tansey, I. P., Spray Technol. Market 4, 26-29 (1994).
Attention has also been given to the design of dry powder aerosol
surface texture, regarding particularly the need to avoid particle
aggregation, a phenomenon which considerably diminishes the
efficiency of inhalation therapies owing to particle aggregation.
French, D. L., Edwards, D. A. and Niven, R. W., J. Aerosol Sci. 27,
769-783 (1996). Attention has not been given to the possibility of
using large particle size (>5 .mu.m) as a means to improve
aerosolization efficiency, despite the fact that intraparticle
adhesion diminishes with increasing particle size. French, D. L.,
Edwards, D. A. and Niven, R. W. J. Aerosol Sci. 27, 769-783 (1996).
This is because particles of standard mass density (mass density
near 1 g/cm.sup.3) and mean diameters >5 .mu.m are known to
deposit excessively in the upper airways or the inhaler device.
Heyder, J. et al., J. Aerosol Sci., 17:811-825 (1986). For this
reason, dry powder aerosols for inhalation therapy are generally
produced with mean diameters primarily in the range of <5 .mu.m.
Ganderton, D., J. Biopharmaceutical Sciences 3:101-105 (1992); and
Gonda, I. "Physico-Chemical Principles in Aerosol Delivery," in
Topics in Pharmaceutical Sciences 1991, Crommelin, D. J. and K. K.
Midha, Eds., Medpharm Scientific Publishers, Stuttgart, pp. 95-115,
1992. Large "carrier" particles (containing no drug) have been
co-delivered with therapeutic aerosols to aid in achieving
efficient aerosolization among other possible benefits. French, D.
L., Edwards, D. A. and Niven, R. W. J. Aerosol Sci. 27, 769-783
(1996).
[0007] Local and systemic inhalation therapies can often benefit
from a relatively slow controlled release of the therapeutic agent.
Gonda, I., "Physico-chemical principles in aerosol delivery," in:
Topics in Pharmaceutical Sciences 1991, D. J. A. Crommelin and K.
K. Midha, Eds., Stuttgart: Medpharm. Scientific Publishers, pp.
95-117, (1992). Slow release from a therapeutic aerosol can prolong
the residence of an administered drug in the airways or acini, and
diminish the rate of drug appearance in the bloodstream. Also,
patient compliance is increased by reducing the frequency of
dosing. Langer, R., Science, 249:1527-1533 (1990); and Gonda, I.
"Aerosols for delivery of therapeutic and diagnostic agents to the
respiratory tract," in Critical Reviews in Therapeutic Drug Carrier
Systems 6:273-313, (1990).
[0008] The human lungs can remove or rapidly degrade hydrolytically
cleavable deposited aerosols over periods ranging from minutes to
hours. In the upper airways, ciliated epithelia contribute to the
"mucociliary escalator" by which particles are swept from the
airways toward the mouth. Pavia, D. "Lung Mucociliary Clearance,"
in Aerosols and the Lung: Clinical and Experimental Aspects,
Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984.
Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989). In
the deep lungs, alveolar macrophages are capable of phagocytosing
particles soon after their deposition. Warheit, M. B. and Hartsky,
M. A., Microscopy Res. Tech. 26:412-422 (1993); Brain, J. D.,
"Physiology and Pathophysiology of Pulmonary Macrophages," in The
Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds.,
Plenum, New York, pp. 315-327, 1985; Dorries, A. M. and Valberg, P.
A., Am. Rev. Resp. Disease 146, 831-837 (1991); and Gehr, P. et al.
Microscopy Res. and Tech., 26, 423-436 (1993). As the diameter of
particles exceeds 3 .mu.m, there is increasingly less phagocytosis
by macrophages. Kawaguchi, H. et al., Biomaterials 7:61-66 (1986);
Krenis, L. J. and Strauss, B., Proc. Soc. Exp. Med., 107:748-750
(1961); and Rudt, S. and Muller, R. H., J. Contr. Rel., 22:263-272
(1992). However, increasing the particle size also minimizes the
probability of particles (possessing standard mass density)
entering the airways and acini due to excessive deposition in the
oropharyngeal or nasal regions. Heyder, J. et al., J. Aerosol Sci.,
17: 811-825 (1986). An effective dry-powder inhalation therapy for
both short and long term release of therapeutics, either for local
or systemic delivery, requires a powder that displays minimum
aggregation, as well as a means of avoiding or suspending the
lung's natural clearance mechanisms until drugs have been
effectively delivered.
[0009] There is a need for improved inhaled aerosols for pulmonary
delivery of therapeutic agents. There is a need for the development
of drug carriers which are capable of delivering the drug in an
effective amount into the airways or the alveolar zone of the lung.
There further is a need for the development of drug carriers for
use as inhaled aerosols which are biodegradable and are capable of
controlled release of drug within the airways or in the alveolar
zone of the lung.
[0010] It is therefore an object of the present invention to
provide improved carriers for the pulmonary delivery of therapeutic
agents. It is a further object of the invention to provide inhaled
aerosols which are effective carriers for delivery of therapeutic
agents to the deep lung. It is another object of the invention to
provide carriers for pulmonary delivery which avoid phagocytosis in
the deep lung. It is a further object of the invention to provide
carriers for pulmonary drug delivery which are capable of
biodegrading and releasing the drug at a controlled rate.
SUMMARY OF THE INVENTION
[0011] Improved aerodynamically light particles for drug delivery
to the pulmonary system, and methods for their synthesis and
administration are provided. In a preferred embodiment, the
particles are made of a biodegradable material, have a tap density
less than 0.4 g/cm.sup.3 and a mean diameter between 5 .mu.m and 30
.mu.m. In one embodiment, for example, at least 90% of the
particles have a mean diameter between 5 .mu.m and 30 .mu.m. The
particles may be formed of biodegradable materials such as
biodegradable polymers, proteins, or other water-soluble materials.
For example, the particles may be formed of a functionalized
polyester graft copolymer consisting of a linear
.alpha.-hydroxy-acid polyester backbone having at least one amino
acid residue incorporated per molecule therein and at least one
poly(amino acid) side chain extending from an amino acid group in
the polyester backbone. Other examples include particles formed of
water-soluble excipients, such as trehalose or lactose, or
proteins, such as lysozyme or insulin. The aerodynamically light
particles can be used for enhanced delivery of a therapeutic agent
to the airways or the alveolar region of the lung. The particles
incorporating a therapeutic agent may be effectively aerosolized
for administration to the respiratory tract to permit systemic or
local delivery of a wide variety of therapeutic agents. They
optionally may be co-delivered with larger carrier particles, not
carrying a therapeutic agent, which have for example a mean
diameter ranging between about 50 .mu.m and 100 .mu.m.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph comparing total particle mass of
aerodynamically light and non-light, control particles deposited on
the nonrespirable and respirable stages of a cascade impactor
following aerosolization.
[0013] FIG. 2 is a graph comparing total particle mass deposited in
the trachea and after the carina (lungs) in rat lungs and upper
airways following intratracheal aerosolization during forced
ventilation of aerodynamically light PLAL-Lys particles and
control, non-light PLA particles.
[0014] FIG. 3 is a graph comparing total particle recovery of
aerodynamically light PLAL-Lys particles and control PLA particles
in rat lungs and following bronchoalveolar lavage.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Aerodynamically light, biodegradable particles for improved
delivery of therapeutic agents to the respiratory tract are
provided. The particles can be used in one embodiment for
controlled systemic or local drug delivery to the respiratory tract
via aerosolization. In a preferred embodiment, the particles have a
tap density less than about 0.4 g/cm.sup.3. Features of the
particle which can contribute to low tap density include irregular
surface texture and porous structure. Administration of the low
density particles to the lung by aerosolization permits deep lung
delivery of relatively large diameter therapeutic aerosols, for
example, greater than 5 .mu.m in mean diameter. A rough surface
texture also can reduce particle agglomeration and provide a highly
flowable powder, which is ideal for aerosolization via dry powder
inhaler devices, leading to lower deposition in the mouth, throat
and inhaler device.
[0016] Density and Size of Aerodynamically Light Particles
[0017] Particle Size
[0018] The mass mean diameter of the particles can be measured
using a Coulter Counter. The aerodynamically light particles are
preferably at least about 5 microns in diameter. The diameter of
particles in a sample will range depending upon depending on
factors such as particle composition and methods of synthesis. The
distribution of size of particles in a sample can be selected to
permit optimal deposition within targeted sites within the
respiratory tract.
[0019] The aerodynamically light particles may be fabricated or
separated, for example by filtration, to provide a particle sample
with a preselected size distribution. For example, greater than
30%, 50%, 70%, or 80% of the particles in a sample can have a
diameter within a selected range of at least 5 .mu.m. The selected
range within which a certain percentage of the particles must fall
may be for example, between about 5 and 30 .mu.m, or optionally
between 5 and 15 .mu.m. In one preferred embodiment, at least a
portion of the particles have a diameter between about 9 and 11
.mu.m. Optionally, the particle sample also can be fabricated
wherein at least 90%, or optionally 95% or 99%, have a diameter
within the selected range. The presence of the higher proportion of
the aerodynamically light, larger diameter (at least about 5 .mu.m)
particles in the particle sample enhances the delivery of
therapeutic or diagnostic agents incorporated therein to the deep
lung.
[0020] In one embodiment, in the particle sample, the interquartile
range may be 2 .mu.m, with a mean diameter for example of 7.5, 8.0,
8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0 or 13.5
.mu.m. Thus, for example, at least 30%, 40%, 50% or 60% of the
particles may have diameters within the selected range 5.5-7.5
.mu.m, 6.0-8.0 .mu.m, 6.5-8.5 .mu.m, 7.0-9.0 .mu.m, 7.5-9.5 .mu.m,
8.0-10.0 .mu.m, 8.5-10.5 .mu.m, 9.0-11.0 .mu.m, 9.5-11.5 .mu.m,
10.0-12.0 .mu.m, 10.5-12.5 .mu.m, 11.0-13.0 .mu.m, 11.5-13.5 .mu.m,
12.0-14.0 .mu.m, 12.5-14.5 .mu.m or 13.0-15.0 .mu.m. Preferably the
said percentages of particles have diameters within a 1 .mu.m
range, for example, 6.0-7.0 .mu.m, 10.0-11.0 .mu.m or 13.0-14.0
.mu.m.
[0021] The aerodynamically light particles incorporating a
therapeutic drug, and having a tap density less than about 0.4
g/cm.sup.3, with mean diameters of at least about 5 .mu.m, are more
capable of escaping inertial and gravitational deposition in the
oropharyngeal region, and are targeted to the airways or the deep
lung. The use of larger particles (mean diameter at least about 5
.mu.m) is advantageous since they are able to aerosolize more
efficiently than smaller, non-light aerosol particles such as those
currently used for inhalation therapies.
[0022] In comparison to smaller non-light particles, the larger (at
least about 5 .mu.m) aerodynamically light particles also can
potentially more successfully avoid phagocytic engulfment by
alveolar macrophages and clearance from the lungs, due to size
exclusion of the particles from the phagocytes' cytosolic space.
Phagocytosis of particles by alveolar macrophages diminishes
precipitously as particle diameter increases beyond 3 .mu.m.
Kawaguchi, H. et al., Biomaterials 7: 61-66 (1986); Krenis, L. J.
and Strauss, B., Proc. Soc. Exp. Med., 107:748-750 (1961); and
Rudt, S. and Muller, R. H., J. Contr. Rel., 22: 263-272 (1992). For
particles of statistically isotropic shape (on average, particles
of the powder possess no distinguishable orientation), such as
spheres with rough surfaces, the particle envelope volume is
approximately equivalent to the volume of cytosolic space required
within a macrophage for complete particle phagocytosis.
[0023] Aerodynamically light particles thus are capable of a longer
term release of a therapeutic agent. Following inhalation,
aerodynamically light biodegradable particles can deposit in the
lungs (due to their relatively low tap density), and subsequently
undergo slow degradation and drug release, without the majority of
the particles being phagocytosed by alveolar macrophages. The drug
can be delivered relatively slowly into the alveolar fluid, and at
a controlled rate into the blood stream, minimizing possible toxic
responses of exposed cells to an excessively high concentration of
the drug. The aerodynamically light particles thus are highly
suitable for inhalation therapies, particularly in controlled
release applications. The preferred mean diameter for
aerodynamically light particles for inhalation therapy is at least
about 5 .mu.m, for example between about 5 and 30 .mu.m.
[0024] The particles may be fabricated with the appropriate
material, surface roughness, diameter and tap density for localized
delivery to selected regions of the respiratory tract such as the
deep lung or upper airways. For example, higher density or larger
particles may be used for upper airway delivery, or a mixture of
different sized particles in a sample, provided with the same or
different therapeutic agent may be administered to target different
regions of the lung in one administration.
[0025] Particle Density and Deposition
[0026] The particles having a diameter of at least about 5 .mu.m
and incorporating a therapeutic or diagnostic agent preferably are
aerodynamically light. As used herein, the phrase "aerodynamically
light particles" refers to particles having a tap density less than
about 0.4 g/cm.sup.3. The tap density of particles of a dry powder
may be obtained using a GeoPyc.TM. (Micrometrics Instrument Corp.,
Norcross, Ga. 30093). Tap density is a standard measure of the
envelope mass density. The envelope mass density of an isotropic
particle is defined as the mass of the particle divided by the
minimum sphere envelope volume within which it can be enclosed.
[0027] Inertial impaction and gravitational settling of aerosols
are predominant deposition mechanisms in the airways and acini of
the lungs during normal breathing conditions. Edwards, D. A., J.
Aerosol Sci. 26:293-317 (1995). The importance of both deposition
mechanisms increases in proportion to the mass of aerosols and not
to particle (or envelope) volume. Since the site of aerosol
deposition in the lungs is determined by the mass of the aerosol
(at least for particles of mean aerodynamic diameter greater than
approximately 1 .mu.m), diminishing the tap density by increasing
particle surface irregularities and particle porosity permits the
delivery of larger particle envelope volumes into the lungs, all
other physical parameters being equal.
[0028] The low tap density particles have a small aerodynamic
diameter in comparison to the actual envelope sphere diameter. The
aerodynamic diameter, d.sub.aer, is related to the envelope sphere
diameter, d (Gonda, I., "Physico-chemical principles in aerosol
delivery," in Topics in Pharmaceutical Sciences 1991 (eds. D. J. A.
Crommelin and K. K. Midha), pp. 95-117, Stuttgart: Medpham
Scientific Publishers, 1992) by the formula:
d.sub.aer=d{square root}{square root over (.rho.)}
[0029] where the envelope mass .rho. is in units of g/cm.sup.3.
Maximal deposition of monodisperse aerosol particles in the
alveolar region of the human lung (.about.60%) occurs for an
aerodynamic diameter of approximately d.sub.aer=3 .mu.m, Heyder, J.
et al., J. Aerosol Sci., 17: 811-825 (1986). Due to their small
envelope mass density, the actual diameter d of aerodynamically
light particles comprising a monodisperse inhaled powder that will
exhibit maximum deep-lung deposition is:
d=3/{square root}{square root over (.rho.)} .mu.m (where .rho.<1
g/cm.sup.3);
[0030] where d is always greater than 3 .mu.m. For example,
aerodynamically light particles that display an envelope mass
density, .rho.=0.1 g/cm.sup.3, will exhibit a maximum deposition
for particles having envelope diameters as large as 9.5 .mu.m. The
increased particle size diminishes interparticle adhesion forces.
Visser, J., Powder Technology, 58:1-10. Thus, large particle size
increases efficiency of aerosolization to the deep lung for
particles of low envelope mass density, in addition to contributing
to lower phagocytic losses.
[0031] Particle Materials
[0032] In order to serve as efficient and safe drug carriers in
drug delivery systems, the aerodynamically light particles
preferably are biodegradable and biocompatible, and optionally are
capable of biodegrading at a controlled rate for delivery of a
drug. The particles can be made of any material which is capable of
forming a particle having a tap density less than about 0.4
g/cm.sup.3. Both inorganic and organic materials can be used. For
example, ceramics may be used. Other non-polymeric materials (e.g.
fatty acids) may be used which are capable of forming
aerodynamically light particles as defined herein. Different
properties of the particle can contribute to the aerodynamic
lightness including the composition forming the particle, and the
presence of irregular surface structure or pores or cavities within
the particle.
[0033] Polymeric Particles
[0034] The particles may be formed from any biocompatible, and
preferably biodegradable polymer, copolymer, or blend, which is
capable of forming particles having a tap density less than about
0.4 g/cm.sup.3.
[0035] Surface eroding polymers such as polyanhydrides may be used
to form the aerodynamically light particles. For example,
polyanhydrides such as poly[(p-carboxyphenoxy)-hexane anhydride]
(PCPH) may be used. Biodegradable polyanhydrides are described, for
example, in U.S. Pat. No. 4,857,311, the disclosure of which is
incorporated herein by reference.
[0036] In another embodiment, bulk eroding polymers such as those
based on polyesters including poly(hydroxy acids) can be used. For
example, polyglycolic acid (PGA) or polylactic acid (PLA) or
copolymers thereof may be used to form the aerodynamically light
particles, wherein the polyester has incorporated therein a charged
or functionalizable group such as an amino acid as described
below.
[0037] Other polymers include polyamides, polycarbonates,
polyalkylenes such as polyethylene, polypropylene, poly(ethylene
glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly
vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and
polyvinyl esters, polymers of acrylic and methacrylic acids,
celluloses and other polysaccharides, and peptides or proteins, or
copolymers or blends thereof which are capable of forming
aerodynamically light particles with a tap density less than about
0.4 g/cm.sup.3. Polymers may be selected with or modified to have
the appropriate stability and degradation rates in vivo for
different controlled drug delivery applications.
[0038] Polyester Graft Copolymers
[0039] In one preferred embodiment, the aerodynamically light
particles are formed from functionalized polyester graft
copolymers, as described in Hrkach et al., Macromolecules,
28:4736-4739 (1995); and Hrkach et al., "Poly(L-Lactic
acid-co-amino acid) Graft Copolymers: A Class of Functional
Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers
for Bioapplications, ACS Symposium Series No. 627, Raphael M.
Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp.
93-101, 1996, the disclosures of which are incorporated herein by
reference. The functionalized graft copolymers are copolymers of
polyesters, such as poly(glycolic acid) or poly(lactic acid), and
another polymer including functionalizable or ionizable groups,
such as a poly(amino acid). In a preferred embodiment, comb-like
graft copolymers are used which include a linear polyester backbone
having amino acids incorporated therein, and poly(amino acid) side
chains which extend from the amino acid residues in the polyester
backbone. The polyesters may be polymers of .alpha.-hydroxy acids
such as lactic acid, glycolic acid, hydroxybutyric acid and hydroxy
valeric acid, or derivatives or combinations thereof. The inclusion
of ionizable side chains, such as polylysine, in the polymer has
been found to enable the formation of more aerodynamically light
particles, using techniques for making microparticles known in the
art, such as solvent evaporation. Other ionizable groups, such as
amino or carboxyl groups, may be incorporated, covalently or
noncovalently, into the polymer to enhance surface roughness and
porosity. For example, polyalanine could be incorporated into the
polymer.
[0040] An exemplary polyester graft copolymer, which may be used to
form aerodynamically light polymeric particles is the graft
copolymer, poly(lactic acid-co-lysine-graft-lysine) (PLAL-Lys),
which has a polyester backbone consisting of poly(L-lactic acid-co-
L-lysine) (PLAL), and grafted poly-lysine chains. PLAL-Lys is a
comb-like graft copolymer having a backbone composition, for
example, of 98 mol % lactic acid and 2 mol % lysine and
poly(lysine) side chains extending from the lysine sites of the
backbone.
[0041] PLAL-Lys may be synthesized as follows. First, the PLAL
copolymer consisting of L-lactic acid units and approximately 1-2%
N .epsilon. carbobenzoxy-L-lysine (Z-L-lysine) units is synthesized
as described in Barrera et al., J. Am. Chem. Soc., 115:11010
(1993). Removal of the Z protecting groups of the randomly
incorporated lysine groups in the polymer chain of PLAL yields the
free .epsilon.-amine which can undergo further chemical
modification. The use of the poly(lactic acid) copolymer is
advantageous since it biodegrades into lactic acid and lysine,
which can be processed by the body. The existing backbone lysine
groups are used as initiating sites for the growth of poly(amino
acid) side chains.
[0042] The lysine .epsilon.-amino groups of linear poly(L-lactic
acid-co-L-lysine) copolymers initiate the ring opening
polymerization of an amino acid N-.epsilon. carboxyanhydride (NCA)
to produce poly(L-lactic acid-co-amino acid) comblike graft
copolymers. In a preferred embodiment, NCAs are synthesized by
reacting the appropriate amino acid with triphosgene. Daly et al.,
Tetrahedron Lett., 29:5859 (1988). The advantage of using
triphosgene over phosgene gas is that it is a solid material, and
therefore, safer and easier to handle. It also is soluble in THF
and hexane so any excess is efficiently separated from the
NCAs.
[0043] The ring opening polymerization of amino acid
N-carboxyanhydrides (NCAs) is initiated by nucleophilic initiators
such as amines, alcohols, and water. The primary amine initiated
ring opening polymerization of NCAs allows good control over the
degree of polymerization when the monomer to initiator ratio (M/I)
is less than 150. Kricheldorf, H. R. in Models of Biopolymers by
Ring-Opening Polymerization, Penczek, S., Ed., CRC Press, Boca
Raton, 1990, Chapter 1; Kricheldorf, H. R.
.alpha.-Aminoacid-N-Carboxy Anhydrides and Related Heterocycles,
Springer-Verlag, Berlin, 1987; and Imanishi, Y. in Ring-Opening
Polymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier,
London, 1984, Volume 2, Chapter 8. Methods for using lysine
.epsilon.-amino groups as polymeric initiators for NCA
polymerizations are described in the art. Sela, M. et al., J. Am.
Chem. Soc., 78: 746 (1956).
[0044] In the reaction of an amino acid NCA with PLAL, the
nucleophilic primary .epsilon.-amino of the lysine side chain
attacks C-5 of the NCA leading to ring opening and formation of the
amino acid amide, along with the evolution of CO.sub.2. Propagation
takes place via further attack of the amino group of the amino acid
amides on subsequent NCA molecules. The degree of polymerization of
the poly(amino acid) side chains, the corresponding amino acid
content in the graft copolymers and their resulting physical and
chemical characteristics can be controlled by changing the M/I
ratio for the NCA polymerization--that is, changing the ratio of
NCA to lysine .epsilon.-amino groups of pLAL. Thus, in the
synthesis, the length of the poly(amino acid), such as
poly(lysine), side chains and the total amino acid content in the
polymer may be designed and synthesized for a particular
application.
[0045] The poly(amino acid) side chains grafted onto or
incorporated into the polyester backbone can include any amino
acid, such as aspartic acid, alanine or lysine, or mixtures
thereof. The functional groups present in the amino acid side
chains, which can be chemically modified, include amino, carboxylic
acid, thiol, guanido, imidazole and hydroxyl groups. As used
herein, the term "amino acid" includes natural and synthetic amino
acids and derivatives thereof. The polymers can be prepared with a
range of amino acid side chain lengths, for example, about 10-100
or more amino acids, and with an overall amino acid content of, for
example, 7-72% or more depending on the reaction conditions. The
grafting of poly(amino acids) from the pLAL backbone may be
conducted in a solvent such as dioxane, DMF, or CH.sub.2Cl.sub.2,
or mixtures thereof. In a preferred embodiment, the reaction is
conducted at room temperature for about 2-4 days in dioxane.
[0046] Alternatively, the aerodynamically light particles for
pulmonary drug delivery may be formed from polymers or blends of
polymers with different polyester/amino acid backbones and grafted
amino acid side chains, For example, poly(lactic
acid-co-lysine-graft-alanine-lysine) (PLAL-Ala-Lys), or a blend of
PLAL-Lys with poly(lactic acid-co-glycolic acid-block-ethylene
oxide) (PLGA-PEG) (PLAL-Lys-PLGA-PEG) may be used.
[0047] In the synthesis, the graft copolymers may be tailored to
optimize different characteristics of the aerodynamically light
particle including: i) interactions between the agent to be
delivered and the copolymer to provide stabilization of the agent
and retention of activity upon delivery; ii) rate of polymer
degradation and, thereby, rate of drug release profiles; iii)
surface characteristics and targeting capabilities via chemical
modification; and iv) particle porosity.
[0048] Formation of Aerodynamically Light Polymeric Particles
[0049] Aerodynamically light polymeric particles may be prepared
using single and double emulsion solvent evaporation, spray drying,
solvent extraction and other methods well known to those of
ordinary skill in the art. The aerodynamically light particles may
be made, for example using methods for making microspheres or
microcapsules known in the art.
[0050] Methods developed for making microspheres for drug delivery
are described in the literature, for example, as described by
Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987);
Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); and
Mathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988), the
teachings of which are incorporated herein. The selection of the
method depends on the polymer selection, the size, external
morphology, and crystallinity that is desired, as described, for
example, by Mathiowitz, et al., Scanning Microscopy 4, 329-340
(1990); Mathiowitz, et, al., J. Appl. Polymer Sci. 45, 125-134
(1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984),
the teachings of which are incorporated herein.
[0051] In solvent evaporation, described for example, in
Mathiowitz, et al., (1990), Benita, and U.S. Pat. No. 4,272,398 to
Jaffe, the polymer is dissolved in a volatile organic solvent, such
as methylene chloride. Several different polymer concentrations can
be used, for example, between 0.05 and 0.20 g/ml. The drug, either
in soluble form or dispersed as fine particles, is added to the
polymer solution, and the mixture is suspended in an aqueous phase
that contains a surface active agent such as poly(vinyl alcohol).
The aqueous phase may be, for example, a concentration of 1%
poly(vinyl alcohol) w/v in distilled water. The resulting emulsion
is stirred until most of the organic solvent evaporates, leaving
solid microspheres, which may be washed with water and dried
overnight in a lyophilizer.
[0052] Microspheres with different sizes (1-1000 microns) and
morphologies can be obtained by this method which is useful for
relatively stable polymers such as polyesters and polystyrene.
However, labile polymers such as polyanhydrides may degrade due to
exposure to water. For these polymers, solvent removal may be
preferred.
[0053] Solvent removal was primarily designed for use with
polyanhydrides. In this method, the drug is dispersed or dissolved
in a solution of a selected polymer in a volatile organic solvent
like methylene chloride. The mixture is then suspended in oil, such
as silicon oil, by stirring, to form an emulsion. Within 24 hours,
the solvent diffuses into the oil phase and the emulsion droplets
harden into solid polymer microspheres. Unlike solvent evaporation,
this method can be used to make microspheres from polymers with
high melting points and a wide range of molecular weights.
Microspheres having a diameter for example between one and 300
microns can be obtained with this procedure.
[0054] Targeting of Particles
[0055] Targeting molecules can be attached to the aerodynamically
light particles via reactive functional groups on the particles.
For example, targeting molecules can be attached to the amino acid
groups of functionalized polyester graft copolymer particles, such
as PLAL-Lys particles. Targeting molecules permit binding
interaction of the particle with specific receptor sites, such as
those within the lungs. The particles can be targeted by attachment
of ligands which specifically or non-specifically bind to
particular targets. Exemplary targeting molecules include
antibodies and fragments thereof including the variable regions,
lectins, and hormones or other organic molecules capable of
specific binding for example to receptors on the surfaces of the
target cells.
[0056] Therapeutic Agents
[0057] Any of a variety of therapeutic, prophylactic or diagnostic
agents can be incorporated within the aerodynamically light
particles. The aerodynamically light particles can be used to
locally or systemically deliver a variety of therapeutic agents to
an animal. Examples include synthetic inorganic and organic
compounds, proteins and peptides, polysaccharides and other sugars,
lipids, and nucleic acid sequences having therapeutic, prophylactic
or diagnostic activities. Nucleic acid sequences include genes,
antisense molecules which bind to complementary DNA to inhibit
transcription, and ribozymes. The agents to be incorporated can
have a variety of biological activities, such as vasoactive agents,
neuroactive agents, hormones, anticoagulants, inimunomodulating
agents, cytotoxic agents, prophylactic agents, antibiotics,
antivirals, antisense, antigens, and antibodies. In some instances,
the proteins may be antibodies or antigens which otherwise would
have to be administered by injection to elicit an appropriate
response. Compounds with a wide range of molecular weight can be
encapsulated, for example, between 100 and 500,000 grams per
mole.
[0058] Proteins are defined as consisting of 100 amino acid
residues or more; peptides are less than 100 amino acid residues.
Unless otherwise stated, the term protein refers to both proteins
and peptides. Examples include insulin and other hormones.
Polysaccharides, such as heparin, can also be administered.
[0059] The aerodynamically light polymeric aerosols are useful as
carriers for a variety of inhalation therapies. They can be used to
encapsulate small and large drugs, release encapsulated drugs over
time periods ranging from hours to months, and withstand extreme
conditions during aerosolization or following deposition in the
lungs that might otherwise harm the encapsulated therapeutic.
[0060] The aerodynamically light particles may include a
therapeutic agent for local delivery within the lung, such as
agents for the treatment of asthma, emphysema, or cystic fibrosis,
or for systemic treatment. For example, genes for the treatment of
diseases such as cystic fibrosis can be administered, as can beta
agonists for asthma. Other specific therapeutic agents include, but
are not limited to, insulin, calcitonin, leuprolide (or LHRH),
G-CSF, parathyroid hormone-related peptide, somatostatin,
testosterone, progesterone, estradiol, nicotine, fentanyl,
norethisterone, clonidine, scopolomine, salicylate, cromolyn
sodium, salmeterol, formeterol, albuterol, and vallium.
[0061] Administration
[0062] The particles including a therapeutic agent may be
administered alone or in any appropriate pharmaceutical carrier,
such as a liquid, for example saline, or a powder, for
administration to the respiratory system. They can be co-delivered
with larger carrier particles, not including a therapeutic agent,
the latter possessing mass mean diameters for example in the range
50 .mu.m-100 .mu.m.
[0063] Aerosol dosage, formulations and delivery systems may be
selected for a particular therapeutic application, as described,
for example, in Gonda, I. "Aerosols for delivery of therapeutic and
diagnostic agents to the respiratory tract," in Critical Reviews in
Therapeutic Drug Carrier Systems, 6:273-313, 1990; and in Moren,
"Aerosol dosage, forms and formulations," in: Aerosols in Medicine.
Principles, Diagnosis and Therapy, Moren, et al., Eds, Esevier,
Amsterdam, 1985, the disclosures of which are incorporated herein
by reference.
[0064] The greater efficiency of aerosolization by aerodynamically
light particles of relatively large size permits more drug to be
delivered than is possible with the same mass of non-light
aerosols. The relatively large size of aerodynamically light
aerosols depositing in the deep lungs also minimizes potential drug
losses caused by particle phagocytosis. The use of aerodynamically
light polymeric aerosols as therapeutic carriers provides the
benefits of biodegradable polymers for controlled release in the
lungs and long-time local action or systemic bioavailability.
Denaturation of macromolecular drugs can be minimized during
aerosolization since macromolecules are contained and protected
within a polymeric shell. Coencapsulation of peptides with
peptidase-inhibitors can minimize peptide enzymatic
degradation.
[0065] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1
Synthesis of Aerodynamically Light Poly[(p-carboxyphenoxy)-hexane
anhydride] ("PCPH") Particles
[0066] Aerodynamically light poly[(p-carboxyphenoxy)-hexane
anhydride] ("PCPH") particles were synthesized as follows. 100 mg
PCPH (MW.about.25,000) was dissolved in 3.0 mL methylene chloride.
To this clear solution was added 5.0 mL, 1% w/v aqueous polyvinyl
alcohol (PVA, MW.about.25,000, 88 mole % hydrolyzed) saturated with
methylene chloride, and the mixture was vortexed (Vortex Genie 2,
Fisher Scientific) at maximum speed for one minute. The resulting
milky-white emulsion was poured into a beaker containing 95 mL 1%
PVA and homogenized (Silverson Homogenizers) at 6000 RPM for one
minute using a 0.75 inch tip. After homogenization, the mixture was
stirred with a magnetic stirring bar and the methylene chloride
quickly extracted from the polymer particles by adding 2 mL
isopropyl alcohol. The mixture was continued to stir for 35 minutes
to allow complete hardening of the microparticles. The hardened
particles were collected by centrifugation and washed several times
with double distilled water. The particles were freeze dried to
obtain a free-flowing powder void of clumps. Yield, 85-90%.
[0067] The mean diameter of this batch was 6.0 .mu.m, however,
particles with mean diameters ranging from a few hundred nanometers
to several millimeters may be made with only slight modifications.
Scanning electron micrograph photos of a typical batch of PCPH
particles showed the particles to be highly porous with irregular
surface shape. The particles have a tap density less than 0.4
g/cm.sup.3.
EXAMPLE 2
Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric and Copolymeric
Particles
[0068] Aerodynamically Light PLAL-Lys Particles
[0069] PLAL-Lys particles were prepared by dissolving 50 mg of the
graft copolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 ml
dichloromethane dropwise. The polymer solution is emulsified in 100
ml of 5% w/v polyvinyl alcohol solution (average molecular weight
25 KDa, 88% hydrolyzed) using a homogenizer (Silverson) at a speed
of approximately 7500 rpm. The resulting dispersion is stirred
using a magnetic stirrer for 1 hour. Following this period, the pH
is brought to 7.0-7.2 by addition of 0.1 N NaOH solution. Stirring
is continued for an additional 2 hours until the methylene chloride
is completely evaporated and the particles hardened. The particles
are then isolated by centrifugation at 4000 rpm (1600 g) for 10
minutes (Sorvall RC-5B). The supernatant is discarded and the
precipitate washed three times with distilled water followed by
centrifugation for 10 minutes at 4000 rpm each time. Finally, the
particles are resuspended in 5 ml of distilled water, the
dispersion frozen in liquid nitrogen, and lyophilized (Labconco
freeze dryer 8) for at least 48 hours. Particle sizing is performed
using a Coulter counter. Average particle mean diameters ranged
from 100 nm to 14 .mu.m, depending upon processing parameters such
as homogenization speed and time. All particles exhibited tap
densities less than 0.4 g/cm.sup.3. Scanning electron micrograph
photos of the particles showed them to be highly porous with
irregular surfaces.
[0070] Aerodynamically Light PLAL-Ala-Lys Particles
[0071] 100 mg of PLAL-Ala-Lys is completely dissolved in 0.4 ml
trifluoroethanol, then 1.0 ml methylene chloride is added dropwise.
The polymer solution is emulsified in 100 ml of 1% w/v polyvinyl
alcohol solution (average molecular weight 25 KDa, 80% hydrolyzed)
using a sonicator (Sonic&Materal VC-250) for 15 seconds at an
output of 40 W. 2 ml of 1% PVA solution is added to the mixture and
it is vortexed at the highest speed for 30 seconds. The mixture is
quickly poured into a beaker containing 100 ml 0.3% PVA solution,
and stirred for three hours allowing evaporation of the methylene
chloride. Scanning electron micrograph photos of the particles
showed them to possess highly irregular surfaces.
[0072] Aerodynamically Light Copolymer Particles
[0073] Polymeric aerodynamically light particles consisting of a
blend of PLAL-Lys and PLGA-PEG were made. 50 mg of the PLGA-PEG
polymer (molecular weight of PEG: 20 KDa, 1:2 weight ratio of
PEG:PLGA, 75:25 lactide:glycolide) was completely dissolved in 1 ml
dichloromethane. 3 mg of poly(lactide-co-lysine)-polylysine graft
copolymer is dissolved in 0.1 ml dimethylsulfoxide and mixed with
the first polymer solution. 0.2 ml of TE buffer, pH 7.6, is
emulsified in the polymer solution by probe sonication
(Sonic&Materal VC-250) for 10 seconds at an output of 40 W. To
this first emulsion, 2 ml of distilled water is added and mixed
using a vortex mixer at 4000 rpm for 60 seconds. The resulting
dispersion is agitated by using a magnetic stirrer for 3 hours
until methylene chloride is completely evaporated and microspheres
formed. The spheres are then isolated by centrifugation at 5000 rpm
for 30 min. The supernatant is discarded, the precipitate washed
three times with distilled water and resuspended in 5 ml of water.
The dispersion is frozen in liquid nitrogen and lyophilized for 48
hours.
[0074] Variables which may be manipulated to alter the size
distribution of the particles include: polymer concentration,
polymer molecular weight, surfactant type (e.g., PVA, PEG, etc.),
surfactant concentration, and mixing intensity. Variables which may
be manipulated to alter the surface shape and porosity of the
particles include: polymer concentration, polymer molecular weight,
rate of methylene chloride extraction by isopropyl alcohol (or
another miscible solvent), volume of isopropyl alcohol added,
inclusion of an inner water phase, volume of inner water phase,
inclusion of salts or other highly water-soluble molecules in the
inner water phase which leak out of the hardening sphere by osmotic
pressure, causing the formation of channels, or pores, in
proportion to their concentration, and surfactant type and
concentration.
[0075] By scanning electron microscopy (SEM), the PLAL-Lys-PLGA-PEG
particles were highly surface rough and porous. The partictes had a
mean particle diameter of 7 .mu.m. The blend of PLAL-Lys with
poly(lactic acid) (PLA) and/or PLGA-PEG copolymers can be adjusted
to adjust particle porosity and size. Additionally, processing
parameters such as homogeruization speed and time can be adjusted.
Neither PLAL, PLA nor PLGA-PEG alone yields an aerodynamically
light structure when prepared by these techniques.
EXAMPLE 3
Synthesis of Spray-Dried Particles
[0076] Aerodynamically Light Particles Containing Polymer and Drug
Soluble in Common Solvent
[0077] Aerodynamically light 50:50 PLGA particles were prepared by
spray drying with testosterone encapsulated within the particles
according to the following procedures. 2.0 g poly
(D,L-lactic-co-glycolic acid) with a molar ratio of 50:50 (PLGA
50:50, Resomer RG503, B.I. Chemicals, Montvale, N.J.) and 0.50 g
testosterone (Sigma Chemical Co., St. Louis, Mo.) are completely
dissolved in 100 mL dichloromethane at room temperature. The
mixture is subsequently spray-dried through a 0.5 mm nozzle at a
flow rate of 5 mL/min using a Buchi laboratory spray-drier (model
190, Buchi, Germany). The flow rate of compressed air is 700 nl/h.
The inlet temperature is set to 30.degree. C. and the outlet
temperature to 25.degree. C. The aspirator is set to achieve a
vacuum of -20 to -25 bar. The yield is 51% and the mean particle
size is approximately 5 .mu.m. Larger particle size can be achieved
by lowering the inlet compressed air flow rate, as well as by
changing other variables. The particles are aerodynamically light,
as determined by a tap density less than or equal to 0.4
g/cm.sup.3. Porosity and surface roughness can be increased by
varying the inlet and outlet temperatures, among other factors.
[0078] Aerodynamically Light Particles Containing Polymer and Drug
in Different Solvents
[0079] Aerodynamically light PLA particles with a model hydrophilic
drug (dextran) were prepared by spray drying using the following
procedure. 2.0 mL of an aqueous 10% w/v FITC-dextran (MW 70,000,
Sigma Chemical Co.) solution was emulsified into 100 mL of a 2% w/v
solution of poly (D,L-lactic acid) (PLA, Resomer R206, B.I.
Chemicals) in dichloromethane by probe sonication (Vibracell
Sonicator, Branson). The emulsion is subsequently spray-dried at a
flow rate of 5 mL/min with an air flow rate of 700 nl/h (inlet
temperature=30.degree. C., outlet temperature=21.degree. C., -20
mbar vacuum). The yield is 56%. The particles are aerodynamically
light, as determined by a tap density of less than 0.4
g/cm.sup.3.
[0080] Aerodynamically Light Protein Particles
[0081] Aerodynamically light lysozyme particles were prepared by
spray drying using the following procedure. 4.75 g lysozyme (Sigma)
was dissolved in 95 mL double distilled water (5% w/v solution) and
spray-dried using a 0.5 mm nozzle and a Buchi laboratory
spray-drier. The flow rate of compressed air was 725 nl/h. The flow
rate of the lysozyme solution was set such that, at a set inlet
temperature of 97-100.degree. C., the outlet temperature is
55-57.degree. C. The aspirator was set to achieve a vacuum of -30
mbar. The enzymatic activity of lysozyme was found to be unaffected
by this process and the yield of the aerodynamically light
particles (tap density less than 0.4 g/cm.sup.3) was 66%.
[0082] Aerodynamically Light High-Molecular Weight Water Soluble
Particles
[0083] Aerodynamically light dextran particles were prepared by
spray drying using the following procedure. 6.04 g DEAE dextran
(Sigma) was dissolved in 242 mL double distilled water (2.5% w/v
solution) and spray-dried using a 0.5 mm nozzle and a Buchi
laboratory spray-drier. The flow rate of compressed air was 750
nl/h. The flow rate of the DEAE-dextran solution was set such that,
at a set inlet temperature of 155.degree. C., the outlet
temperature was 80.degree. C. The aspirator was set to achieve a
vacuum of -20 mbar. The yield of the aerodynamically light
particles (tap density less than 0.4 g/cm.sup.3) was 66% and the
size range ranged between 1-15 .mu.m.
[0084] Aerodynamically Light Low-Molecular Weight Water-Soluble
Particles
[0085] Aerodynamically light trehalose particles were prepared by
spray drying using the following procedure. 4.9 g trehalose (Sigma)
was dissolved in 192 mL double distilled water (2.5% w/v solution)
and spray-dried using a 0.5 mm nozzle and a Buchi laboratory
spray-drier. The flow rate of compressed air was 650 nl/h. The flow
rate of the trehalose solution was set such that, at a set inlet
temperature of 100.degree. C., the outlet temperature was
60.degree. C. The aspirator was set to achieve a vacuum of -30
mbar. The yield of the aerodynamically light particles (tap density
less than 0.4 g/cm.sup.3) was 36% and the size range ranged between
1-15 .mu.m.
[0086] Aerodynamically Light Low-Molecular Weight Water-Soluble
Particles
[0087] Polyethylene glycol (PEG) is a water-soluble macromolecule,
however, it cannot be spray dried from an aqueous solution since it
melts at room temperatures below that needed to evaporate water. As
a result, we have spray-dried PEG at low temperatures from a
solution in dichloromethane, a low boiling organic solvent.
Aerodynamically light PEG particles were prepared by spray drying
using the following procedure. 5.0 g PEG (MW 15,000-20,000, Sigma)
was dissolved in 100 mL double distilled water (5.0% w/v solution)
and spray-dried using a 0.5 mm nozzle and a Buchi laboratory
spray-drier. The flow rate of compressed air 750 nl/h. The flow
rate of the PEG solution was set such that, at a set inlet
temperature of 45.degree. C., the outlet temperature was
34-35.degree. C. The aspirator was set to achieve a vacuum of -22
mbar. The yield of the aerodynamically light particles (tap density
less than 0.4 g/cm.sup.3) was 67% and the size range ranged between
1-15 .mu.m.
EXAMPLE 4
Rhodarnine Isothiocyanate Labeling of PLAL and PLAL-Lys
Particles
[0088] Aerodynamically light particles were compared with control
particles, referred to herein as "non-light" particles. Lysine
amine groups on the surface of aerodynamically light (PLAL-Lys) and
control, non-light (PLAL) particles, with similar mean diameters
(6-7 .mu.m) and size distributions (standard deviations 3-4 .mu.m)
were labeled with Rhodamine isothiocyanate. The tap density of the
porous PLAL-Lys particles was 0.1 g/cm.sup.3 and that of the
non-light PLAL particles was 0.8 g/cm.sup.3.
[0089] The rhodamine-labeled particles were characterized by
confocal microscopy. A limited number of lysine functionalities on
the surface of the solid particle were able to react with rhodamine
isothiocyanate, as evidenced by the fluorescent image. In the
aerodynamically light particle, the higher lysine content in the
graft copolymer and the porous particle structure result in a
higher level of rhodamine attachment, with rhodamine attachment
dispersed throughout the intersticies of the porous structure. This
also demonstrates that targeting molecules can be attached to the
aerodynamically light particles for interaction with specific
receptor sites within the lungs via chemical attachment of
appropriate targeting agents to the particle surface.
EXAMPLE 5
Aerosolization of PLAL and PLAL-Lys Particles
[0090] To determine whether large aerodynamically light particles
can escape (mouth, throat and inhaler) deposition and more
efficiently enter the airways and acini than nonporous particles of
similar size (referred to herein as nonlight or control particles),
aerosolization and deposition of aerodynamically light PLAL-Lys
(mean diameter 6.3 .mu.m) or control, non-light PLAL (mean diameter
6.9 .mu.m) particles were examined in vitro using a cascade
impactor system.
[0091] 20 mg of the aerodynamically light or non-light
microparticles were placed in gelatine capsules (Eli Lilly), the
capsules loaded into a Spinhaler dry powder inhaler (DPI) (Fisons),
and the DPI activated. Particles were acrosolized into a Mark I
Andersen Impactor (Andersen Samplers, GA) from the DPI for 30
seconds at 28.3 l/min flow rate. Each plate of the Andersen
Impactor was previously coated with Tween 80 by immersing the
plates in an acetone solution (5% w/vol) and subsequently
evaporating the acetone in a oven at 60.degree. C. for 5 min. After
aerosolization and deposition, particles were collected from each
stage of the impactor system in separate volumetric flasks by
rinsing each stage with a NaOH solution (0.2 N) in order to
completely degrade the polymers. After incubation at 37.degree. C.
for 12 h, the fluorescence of each solution was measured
(wavelengths of 554 nm excitation, 574 nm emission).
[0092] Particles were determined as nonrespirable (mean aerodynamic
diameter exceeding 4.7 .mu.m: impactor estimate) if they deposited
on the first three stages of the impactor, and respirable (mean
aerodynamic diameter 4.7 .mu.m or less) if they deposited on
subsequent stages. FIG. 1 shows that less than 10% of the non-light
(PLAL) particles that exit the DPI are respirable. This is
consistent with the large size of the microparticles and their
standard mass density. On the other hand, greater than 55% of the
aerodynamically light (PLAL-Lys) particles are respirable, even
though the geometrical dimensions of the two particle types are
almost identical. The lower tap density of the aerodynamically
light (PLAL-Lys) microparticles is responsible for this improvement
in particle penetration, as discussed further below.
[0093] The non-light (PLAL) particles also inefficiently aerosolize
from the DPI; typically, less than 40% of the non-light particles
exited the Spinhaler DPI for the protocol used. The aerodynamically
light (PLAL-Lys) particles exhibited much more efficient
aerosolization (approximately 80% of the aerodynamically light
microparticles typically exited the DPI during aerosolization).
[0094] The combined effects of efficient aerosolization and high
respirable fraction of aerosolized particle mass means that a far
greater fraction of an aerodynamically light particle powder is
likely to deposit in the lungs than of a non-light particle
powder.
EXAMPLE 6
In Vivo Aerosolization of PLAL and PLAL-lys Particles
[0095] The penetration of aerodynamically light and non-light
polymeric PLAL-Lys and PLAL microparticles into the lungs was
evaluated in an in vivo experiment involving the aerosolization of
the microparticles into the airways of live rats.
[0096] Male Spraque Dawley rats (150-200 g) were anesthetized using
ketamine (90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was
placed ventral side up on a surgical table provided with a
temperature controlled pad to maintain physiological temperature.
The animal was cannulated above the carina with an endotracheal
tube connected to a Harvard ventilator. The animal was force
ventilated for 20 minutes at 300 ml/min. 50 mg of aerodynamically
light (PLAL-Lys) or non-light (PLA) microparticles were introduced
into the endotracheal tube.
[0097] Following the period of forced ventilation, the animal was
euthanized and the lungs and trachea were separately washed using
bronchoalveolar lavage. A tracheal cannula was inserted, tied into
place, and the air-ways were washed with 10 ml aliquots of HBSS.
The lavage procedure was repeated until a total volume of 30 ml was
collected. The lavage fluid was centrifuged (400 g) and the pellets
collected and resuspended in 2 ml of phenol red-free Hanks balanced
salt solution (Gibco, Grand Island, N.Y.) without Ca.sup.2+ and
Mg.sup.2+ (HBSS). 100 ml were removed for particle counting using a
hemacytometer. The remaining solution was mixed with 10 ml of 0.4 N
NaOH. After incubation at 37.degree. C. for 12 h, the fluorescence
of each solution was measured (wavelengths of 554 nm excitation,
574 nm emission).
[0098] FIG. 2 is a bar graph showing total particle mass deposited
in the trachea and after the carina (lungs) in rat lungs and upper
airways following intratracheal aerosolization during forced
ventilation. The PLAL-Lys aerodynamically light particles had a
mean diameter 6.9 .mu.m. The non-light PLAL particles had a mean
diameter of 6.7 .mu.m. Percent tracheal aerodynamically light
particle deposition was 54.5, and non-light deposition was 77.0.
Percent aerodynamically light particle deposition in the lungs was
46.8 and non-light deposition was 23.0.
[0099] The non-light (PLAL) particles deposited primarily in the
trachea (approximately 79% of all particle mass that entered the
trachea). This result is similar to the in vitro performance of the
non-light microparticles and is consistent with the relatively
large size of the nonlight particles. Approximately 54% of the
aerodynamically light (PLAL-Lys) particle mass deposited in the
trachea. Therefore, about half of the aerodynamically light
particle mass that enters the trachea traverses through the trachea
and into the airways and acirii of the rat lungs, demonstrating the
effective penetration of the aerodynamically light particles into
the lungs.
[0100] Following bronchoalveolar lavage, particles remaining in the
rat lungs were obtained by careful dissection of the individual
lobes of the lungs. The lobes were placed in separate petri dishes
containing 5 ml of HBSS. Each lobe was teased through 60 mesh
screen to dissociate the tissue and was then filtered through
cotton gauze to remove tissue debris and connective tissue. The
petri dish and gauze were washed with an additional 15 ml of HBSS
to maximize microparticle collection. Each tissue preparation was
centrifuged and resuspended in 2 ml of HBSS and the number of
particles counted in a hemacytometer. The particle numbers
remaining in the lungs following the bronchoalveolar lavage are
shown in FIG. 3. Lobe numbers correspond to: 1) left lung, 2)
anterior, 3) median, 4) posterior, 5) postcaval. A considerably
greater number of aerodynamically light PLAL-Lys particles enters
every lobe of the lungs than the nonlight PLAL particles, even
though the geometrical dimensions of the two types of particles are
essentially the same. These results reflect both the efficiency of
aerodynamically light particle aerosolization and the propensity of
the aerodynamically light particles to escape deposition prior to
the carina or first bifurcation.
[0101] Modifications and variations of the present invention will
be obvious to those skilled in the art from the foregoing detailed
description. Such modifications and variations are intended to come
within the scope of the following claims.
* * * * *